research papers
274
doi:10.1107/S1399004714025243
ActaCryst.
(2015). D
71
, 274–282
Acta Crystallographica Section D
Biological
Crystallography
ISSN 1399-0047
Nitrogenase MoFe protein from
Clostridium
pasteurianum
at 1.08 A
̊
resolution: comparison with
the
Azotobacter vinelandii
MoFe protein
Li-Mei Zhang,
a
Christine N.
Morrison,
a
Jens T. Kaiser
a
and
Douglas C. Rees
a,b
*
a
Division of Chemistry and Chemical
Engineering, California Institute of Technology,
Pasadena, CA 91125, USA, and
b
Howard
Hughes Medical Institute, California Institute of
Technology, Pasadena, CA 91125, USA
Correspondence e-mail: dcrees@caltech.edu
The X-ray crystal structure of the nitrogenase MoFe protein
from
Clostridium pasteurianum
(Cp1) has been determined at
1.08 A
̊
resolution by multiwavelength anomalous diffraction
phasing. Cp1 and the ortholog from
Azotobacter vinelandii
(Av1) represent two distinct families of nitrogenases, differing
primarily by a long insertion in the
-subunit and a deletion in
the
-subunit of Cp1 relative to Av1. Comparison of these
two MoFe protein structures at atomic resolution reveals
conserved structural arrangements that are significant to the
function of nitrogenase. The FeMo cofactors defining the
active sites of the MoFe protein are essentially identical
between the two proteins. The surrounding environment is
also highly conserved, suggesting that this structural arrange-
ment is crucial for nitrogen reduction. The P clusters are
likewise similar, although the surrounding protein and solvent
environment is less conserved relative to that of the FeMo
cofactor. The P cluster and FeMo cofactor in Av1 and Cp1 are
connected through a conserved water tunnel surrounded by
similar secondary-structure elements. The long
-subunit
insertion loop occludes the presumed Fe protein docking
surface on Cp1 with few contacts to the remainder of the
protein. This makes it plausible that this loop is repositioned
to open up the Fe protein docking surface for complex
formation.
Received 27 October 2014
Accepted 17 November 2014
PDB reference:
MoFe
protein, 4wes
1. Introduction
Biological nitrogen fixation is the process of reducing atmos-
pheric dinitrogen to a biologically available form, such as
ammonia, catalyzed by the enzyme nitrogenase found in
certain bacteria and archaea (Burgess & Lowe, 1996; Howard
& Rees, 2006; Seefeldt
et al.
, 2009; Hu & Ribbe, 2010). This
reaction is powered by adenosine triphosphate (ATP)
hydrolysis, with the requisite electrons supplied from ferre-
doxin or flavodoxin. Nitrogenase is composed of two metal-
loprotein components, the molybdenum–iron (MoFe) protein
and the iron (Fe) protein in the well characterized molyb-
denum nitrogenase system. The MoFe protein forms a dimer
of heterodimers (
2
2
) containing two complex metalloclus-
ters in each
heterodimer: the [8Fe–7S] P cluster bridging
the
-subunit and
-subunit and the [7Fe–Mo–9S–C–homo-
citrate] FeMo cofactor buried inside the
-subunit. The Fe
protein is a
2
dimer with one [4Fe–4S] cluster between the
two monomers. During nitrogen fixation, the Fe protein forms
a transient complex with the MoFe protein and transfers
electrons to the MoFe protein with concomitant hydrolysis of
ATP. The FeMo cofactor provides the active site for substrate
reduction, while the P cluster is believed to serve as a relay for
electron transfer from the [4Fe–4S] cluster of the Fe protein to
the FeMo cofactor.
While nitrogenase MoFe proteins are highly conserved
among the nitrogen-fixing species examined so far, differences
do exist. The
Azotobacter vinelandii
MoFe protein (Av1) and
the
Clostridium pasteurianum
MoFe protein (Cp1) have been
recognized as representatives of two distinct groups: group I
and group II, respectively (Wang
et al.
, 1988; Howard
et al.
,
2013). Cp1 and Av1 share the lowest sequence identity
(
37%) among the well studied MoFe protein homologs. In
particular, the group II MoFe proteins represented by Cp1
have a long insertion in the C-terminus of the
-subunit and a
long N-terminal deletion in the
-subunit compared with the
group I proteins represented by Av1 (Howard
et al.
, 2013).
Correlated with the structural differences, Cp1 and Av1 show
distinct physicochemical properties (such as midpoint redox
potentials and optimal pH for enzyme activity; O’Donnell &
Smith, 1978; Kim
et al.
, 1993). One of the most significant
differences is that, in contrast to other MoFe proteins, Cp1
rarely exhibits promiscuity towards orthologous Fe proteins
from other bacterial species, while the
C. pasteurianum
Fe
protein (Cp2) forms an inactive complex with Av1 (Emerich &
Burris, 1978). Considering these differences between Cp1 and
Av1 in sequence, structure and certain properties, the struc-
tural arrangements conserved in both proteins are likely to be
essential for the nitrogenase activity. Therefore, the identifi-
cation of the structural motifs that are conserved between Cp1
and Av1 will render insights into the functional mechanism of
nitrogenase.
A structure of Cp1 has previously only been reported at 3 A
̊
resolution (PDB entry 1mio; Kim
et al.
, 1993). While a 2.2 A
̊
resolution Cp1 structure has been determined, the refined
structure has not been deposited in the PDB (Bolin
et al.
,
1993). In contrast, Av1 has been characterized at atomic
resolution [1.16 A
̊
resolution (PDB entry 1m1n) and 1.0 A
̊
resolution (PDB entry 3u7q) ; Spatzal
et al.
, 2011; Einsle
et al.
,
2002]. Additionally, the
Klebsiella pneumoniae
MoFe protein
(Kp1), which is closely related to Av1 with
70% sequence
identity, has been characterized at 1.6 A
̊
resolution in different
oxidation states (PDB entries 1qh1 in the reduced state, 1qh8
in the oxidized state and 1qgu in the mixed oxidation state;
Mayer
et al.
, 1999). We report in this paper the crystal struc-
ture of Cp1 determined at 1.08 A
̊
resolution, and describe the
three-dimensional structure alignment against the 1.0 A
̊
resolution Av1 structure, with a focus on the structures of
the two metalloclusters and their surrounding environment.
Unless specified elsewhere, the 1.08 A
̊
resolution Cp1 (this
work) and the 1.0 A
̊
resolution Av1 (PDB entry 3u7q) struc-
tures are used in structural comparison between the Cp1 and
Av1 proteins. The mechanistic implications of the similarities
and differences between the Cp1, Av1 and Kp1 proteins are
also discussed.
2. Materials and methods
2.1. Protein purification and crystallization
Purification of Cp1 was performed as described previously
(Kim
et al.
, 1993). Cp1 was crystallized using sitting-drop
vapor diffusion with a reservoir solution consisting of 0.2
M
lithium citrate, 20%(
w
/
v
) polyethylene glycol (PEG) 3350 at
room temperature (
295 K) in a Coy anaerobic chamber.
The Cp1 crystals grown under this crystallization condition
belonged to space group
P
2
1
, with unit-cell parameters
a
= 72.7,
b
= 170.6,
c
= 87.5 A
̊
,
= 91.6
. The crystals were
soaked in precipitation solution containing 10% 2-methyl-2,4-
pentanediol (MPD) and 5 m
M
sodium dithionite for 15 min
before flash-cooling them in liquid nitrogen.
2.2. Data collection and processing
Crystallographic diffraction data were collected on beam-
line 12-2 at the Stanford Synchrotron Radiation Lightsource
(SSRL) with a PILATUS 6M pixel-array detector. The 1.08 A
̊
resolution diffraction data set was collected at 14 000 eV using
an oscillation angle of 0.15
. Full sets of anomalous diffraction
data for experimental phasing were collected at 7117 and
7130 eV, energies corresponding to the inflection point and
above the Fe
K
-edge absorption edge, respectively, with an
oscillation angle of 0.15
and using standard and inverse-beam
modes of data collection. For site-specific X-ray absorption
spectroscopy (Einsle
et al.
, 2007), multiwavelength anomalous
diffraction (MAD) data were collected at different energies
across the Fe
K
-edge absorption edge with an oscillation angle
of 0.5
and an overall oscillation range of 360
at each energy
point. A reference set of 360 diffraction images was collected
at 12 658 eV with an oscillation angle of 0.5
.
The crystallographic data were processed using the
XDS
program package (Kabsch, 2010) and the
CCP
4 program
package (Winn
et al.
, 2011). The phasing information was
obtained from the MAD data (7117 and 7130 eV at the
inflection and above the Fe
K
-edge absorption edge, respec-
tively) using
SHELXC
/
D
/
E
(Sheldrick, 2008). The model was
built manually with
Coot
, using the electron-density map from
experimental phasing and using the protein sequence reported
earlier as a guide (Kim
et al.
, 1993; Emsley
et al.
, 2010). The
research papers
ActaCryst.
(2015). D
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Zhang
etal.
Nitrogenase MoFe protein
275
Table 1
Summary of the data-processing statistics.
Values in parentheses are for the outer shell.
14 000 eV†
(0.886 A
̊
)
7130 eV†
(1.734 A
̊
)
7117 eV†
(1.742 A
̊
)
Space group
P
2
1
Unit-cell parameters
(A
̊
,
)
a
= 72.70,
b
= 170.58,
c
= 87.54,
= 91.63
Resolution (A
̊
)
38.9–1.08
(1.14–1.08)
42.67–2.04
(2.17–2.04)
42.67–2.04
(2.17–2.04)
R
merge
(%)
6.4 (80.3)
2.9 (5.4)
2.7 (4.9)
R
p.i.m.
(%)
3.9 (49.4)
1.7 (3.4)
1.3 (2.3)
Unique reflections
842587 (117365) 124980 (15125) 120979 (14139)
Multiplicity
3.6 (3.6)
6.8 (6.4)
6.8 (6.4)
Completeness (%)
93.0 (88.7)
92.5 (76.8)
91.4 (73.3)
h
I
/
(
I
)
i
9.7 (1.6)
42.8 (22.9)
46.8 (25.6)
Anomalous completeness
(%)
—
90.1 (73.2)
91.4 (73.3)
Anomalous multiplicity —
3.5 (3.4)
3.5 (3.4)
† The X-ray energies (wavelengths) at which the diffraction data were collected.
structure was further refined using
REFMAC
5 (Murshudov
et
al.
, 2011). The models for the metalloclusters in Cp1 were built
into the crystallographic structure based on the location of the
peaks in the positive
F
o
F
c
difference density map and the
anomalous density map. The models were then refined either
without any restraints or using restraints generated from the
1.0 A
̊
resolution Av1 structure (Spatzal
et al.
, 2011). These two
refinement approaches resulted in essentially identical
metalloclusters. The data-collection and refinement statistics
are summarized in Tables 1 and 2. The electron-density
analysis was carried out using in-house software. The structure
validation analysis was performed using
MolProbity
v.4.02
(Chen
et al.
, 2010). The intermolecular contacts were analyzed
using the
CCP
4 program
CONTACT
(Winn
et al.
, 2011).
PyMOL
was used to prepare the figures (DeLano, 2002).
The Fe
K
-edge site-specific X-ray absorption spectroscopy
data were processed as described previously (Einsle
et al.
,
2007). Molybdenum and sulfur were also included in the
anomalous refinement procedure owing to their significant
anomalous signal in the Fe
K
-edge region; since the data sets
were collected at energies remote from the S
K
absorption
edge, a single set of
f
0
and
f
00
parameters was used for all S
atoms.
2.3. Coordinates
Atomic coordinates and structure factors have been
deposited in the Research Collaboratory for Structural
Bioinformatics Protein Data Bank as entry 4wes.
3. Results and discussion
3.1. Overall structure
The 1.08 A
̊
resolution X-ray crystal structure of Cp1 was
determined by experimental phasing using MAD data (see
x
2
for details). The crystal form of Cp1 is similar to the previously
reported cesium-derivative Cp1 structure in space group
P
2
1
,
with unit-cell parameters
a
=72.7,
b
= 170.6,
c
= 87.5 A
̊
,
=
91.6
(Kim
et al.
, 1993). The data-processing and refinement
statistics are summarized in Tables 1 and 2, respectively. An
overview of the Cp1 tetramer structure is shown in Fig. 1. A
total of 1953 residues (out of 1982 residues) and 2484 water
molecules were included in the model for structural refine-
ment. The first two residues in the N-terminus and the last 11–
12 residues in the C-terminus of the
-subunit were not
included in the model owing to weak electron density. The
overall coordinate error in the model is estimated to be
0.022 A
̊
from the diffraction-component precision index
(Cruickshank, 1999). A single nonproline
cis
-peptide bond,
first reported in Kp1 between Trp
251 and Ser
252, is also
present in Cp1 and Av1 (Leu
240–Thr
241 in Cp1 and
Trp
253–Ser
254 in Av1; Mayer
et al.
, 1999; Spatzal
et al.
,
2011). This pair of residues is located in a
-sheet of the
second domain in the
-subunit (Kim & Rees, 1992), about
9A
̊
away from the FeMo cofactor. A few registry errors were
corrected from the previously reported 3.0 A
̊
resolution Cp1
structure, among which the longest (residues 412–420) is
within the
-subunit insertion sequence characteristic of the
group II MoFe proteins (Supplementary Fig. S1; Kim
et al.
,
1993).
With the exceptions of the long insertion (residues 376–429
in Cp1) and deletion (residues 1–47 in Av1) in the
-subunit
and
-subunit, respectively, the overall structures of the Cp1
and Av1 folds are similar and can be superimposed with a
root-mean-square deviation (r.m.s.d.) of the main chain of
0.92 A
̊
for 1554 aligned residues (78% of the Cp1 sequence;
Supplementary Fig. S2). For comparison, the overall r.m.s.d.
deviation is 0.46 A
̊
between the two closely related MoFe
proteins Av1 and Kp1 over 1976 residues (99% of the Kp1
sequence). In addition to these
50-residue insertion/deletion
regions, shorter gaps are present around residues 202–207 in
the
-subunit and residues 162–168 in the
-subunit of Cp1
(both of which correspond to residues
210–220 in Av1).
3.2. Insertion sequence and the Fe protein docking site
The long insert (residues 376–429) in the
-subunit of Cp1
adopts an irregularly structured loop involving residues
386–408 that is flanked by a short helix on each end
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276
Zhang
etal.
Nitrogenase MoFe protein
ActaCryst.
(2015). D
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Table 2
Summary of refinement statistics.
Resolution (A
̊
)
38.9–1.08
R
work
(%)
11.0
R
free
(%)
13.3
R.m.s.d., bonds (A
̊
)
0.012
R.m.s.d., angles (
)
1.64
Average
B
factor (A
̊
2
)
Protein and ligands
15.3
Solvent
33.7
Diffraction precision index (A
̊
)
0.022
Ramachandran statistics (%)
Favored
97.3
Allowed
2.55
Outliers
0.15
Missing residues (residue Nos.)
-Subunit
1, 2, 522–533
0
-Subunit
1, 2, 521–533
Figure 1
Ribbon representation of the Cp1 tetramer viewed down the molecular
(noncrystallographic) twofold symmetry axis. The
-subunits are shaded
in pale cyan and teal, except for the long insertion loops (residues
376–
429), which are highlighted in blue. The
-subunits are colored split pea
and pale green. The FeMo cofactors and the P clusters are depicted as
sticks, with C atoms shown in light gray, N atoms in blue, O atoms in red, S
atoms in yellow, Fe atoms in orange and Mo atoms in cyan.
(Supplementary Fig. S3). As it is positioned on the surface
adjacent to the FeMo cofactor and close to the binding site on
Av1 for the Fe protein, it has been speculated that this
insertion may prevent Cp1 from forming an active complex
with any orthologous Fe proteins (Kim
et al.
, 1993). Indeed,
when superimposing the Cp1 and Cp2 (PDB entry 1cp2)
structures onto different structures of the Av1–Av2 nitro-
genase complex (PDB entries 1n2c, 2afk and 2afh; Tezcan
et
al.
, 2005; Schindelin
et al.
, 1997; Schlessman
et al.
, 1998), the
presumed Fe protein docking site is occluded by residues 386–
392 in Cp1. This suggests that the interaction between Cp1 and
Cp2 involves either a different Fe protein footprint or that the
insertion loop must rearrange. The insertion sequence forms
relatively few contacts with the rest of the protein, primarily
mediated by residues in the
386–
408 region (Supplemen-
tary Table S1). These contacts can be grouped into three
clusters, as shown in Supplementary Fig. S3. Additionally,
intermolecular contacts are observed between residues in the
insertion sequence and residues from adjacent molecules in
the crystal lattice. This suggests that the insertion sequence
may be relatively dynamic in solution, and thus may give the
opportunity for Cp2 binding to Cp1 at the well conserved Fe
protein docking site designated in the Av1–Av2 complex,
while the conformation observed in the crystallographic
structure may be stabilized as a result of crystal packing.
3.3. Structure of the metalloclusters
3.3.1. The FeMo cofactor and surrounding environment
.A
significant advantage of atomic resolution crystal structures is
to minimize the influence of series-termination effects in
Fourier maps, which are of particular concern for the inter-
stitial ligand of the FeMo cofactor, as it is surrounded by six
equidistant irons (Spatzal
et al.
, 2011; Einsle
et al.
, 2002). In the
2
F
o
F
c
map of the 1.08 A
̊
resolution Cp1 structure, the
electron density at the center of the FeMo cofactor clearly
indicates the presence of an interstitial ligand in the cofactor
of Cp1 (Fig. 2
a
). Using the electron-density analyses devel-
oped previously (Spatzal
et al.
, 2011), the interstitial ligand has
similar properties in both Cp1 and Av1, consistent with the
assignment of this atom as carbon. Comparison of the aver-
aged electron density
(
r
) calculated within spheres of
different radii around a given atom type shows that the
interstitial ligand in the FeMo cofactor most closely resembles
proteinaceous carbon, but not nitrogen or oxygen (Fig. 2
b
).
The deviation of
(
r
) for the interstitial ligand from that of
research papers
ActaCryst.
(2015). D
71
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Zhang
etal.
Nitrogenase MoFe protein
277
Figure 2
Characterization of the interstitial ligand in the Cp1 FeMo cofactor. (
a
) The refined crystallographic structure of the FeMo cofactor with the
superimposed 2
F
o
F
c
electron-density map highlighted in light blue and contoured at 3
. The interstitial ligand is modeled as carbon and labeled CX. C
atoms are shown in light gray, N atoms in blue, O atoms in red, S atoms in yellow, Fe atoms in orange and Mo atoms in cyan. Homocitrate is labeled HCA.
(
b
) The averaged electron density
(
r
) of the two crystallographically independent interstitial ligands CX (green) calculated within spheres of the
indicated radii and compared with those calculated for proteinaceous C (gray), N (blue) and O (red) atoms with an isotropic
B
factor no greater than
30 A
̊
2
.(
c
) The variation in electron density (
0
) at the atomic center as a function of the isotropic
B
factor for proteinaceous carbon (gray), nitrogen
(blue) and oxygen (red). Atoms with isotropic
B
factors of <30 A
̊
2
were included in the calculation. The data points representing two crystallographically
independent interstitial ligands CX are shown in green.
Table 3
Comparison of the metal–ligand and metal
metal distances in the FeMo
cofactors of Cp1 and Av1.
Cp1
Av1
Fe—S
2.25
0.03
2.24
0.03
Fe—C
2.00
0.02
2.00
0.01
Mo—S
2.36
0.01
2.36
0.01
Mo—N
2.35
0.01
2.29
0.02
Mo—O
2.20
0.03
2.19
0.02
Short Fe
Fe†
2.64
0.04
2.63
0.03
Long Fe
Fe‡
3.69
0.01
3.70
0.01
Fe1
Fe
Fe
4.97
0.01
5.00
0.01
Fe
Mo
2.68
0.01
2.69
0.03
Fe
Fe
Mo
5.04
0.01
5.06
0.02
Fe1
Fe
Fe
Mo
6.95
0.00
7.00
0.00
† The distances between Fe within each half of the FeMo cofactor (
i.e.
Fe
Fe distances
involving Fe1–Fe4 and Fe
Fe distances involving Fe5–Fe7) and between the belt Fe
atoms located in the two distinct halves of the FeMo cofactor, with the Fe
Fe vector
parallel to the Fe1
Mo axis (
i.e.
Fe2 and Fe6, Fe3 and Fe7, and Fe4 and Fe5). ‡ The
distances between the belt Fe atoms located in the two different halves of the FeMo
cofactor, with the Fe
Fe vector not parallel to the Fe1
Mo axis (
i.e.
between Fe2 and
Fe5/7, between Fe3 and Fe5/6, and between Fe4 and Fe6/7).
proteinaceous carbon at larger radius (>0.8 A
̊
) may reflect the
truncation error caused by the surrounding heavy atoms, such
as Fe atoms, which are about 2 A
̊
away from the interstitial
ligand. In addition, the correlation between the electron
density at the center of a given type of atom and the isotropic
B
factor of the interstitial ligand also falls in the range of
proteinaceous carbon, but not nitrogen or oxygen (Fig. 2
c
).
As summarized in Table 3 (and detailed in Supplementary
Table S2), the metal–ligand and metal
metal distances of the
FeMo cofactor in Cp1 and Av1 are essentially the same. The
largest difference is observed between the C1 carboxyl groups
of the homocitrate (Fig. 3), which leads to O2 of the homo-
citrate in Cp1 being about 0.3 A
̊
closer to Fe6 of the FeMo
cofactor. Interestingly, a more significant displacement (0.7 A
̊
away from Fe6) in the C1 carboxyl group of the homocitrate
has been reported recently in Av1 at high pH, and the C1 arm
has been proposed to possibly play a role in proton transfer
(Howard & Rees, 1994; Yang
et al.
, 2014).
Residues making side-chain contacts to the FeMo cofactor
are highly conserved in all groups of nitrogenases (Howard
et
al.
, 2013). Among these residues, those potentially forming
hydrogen bonds or other polar contacts with the cofactor
through either the peptide backbone or side chains are high-
lighted in Fig. 3. Of the five residues with side chains mediating
polar interactions, four are invariant in both the group I and
group II MoFe proteins (Howard
et al.
, 2013). The fifth
residue, Lys
466 (Lys
426 in Av1), is strictly conserved in
group I and is a dominant single variant with an Arg found in
two cases in group II. The strict conservation of these residues
emphasizes their important roles for the function of nitro-
genase.
A subtle, but intriguing, difference between Cp1 and Av1 is
observed for residue Arg
87 (Arg
96 in Av1) in proximity to
the FeMo cofactor. Arg
87 is conserved with a single variant
(Lys) in all groups of MoFe proteins. In both high-resolution
Av1 structures this Arg consistently shows a significant non-
planar distortion of the guanidinium group, characterized by
an averaged CD—NE—CZ—NH2 torsion angle of around 26
(Spatzal
et al.
, 2011; Einsle
et al.
, 2002). The equivalent torsion
angle in Cp1 is
6
, indicating a more planar group. As a
consequence, there are slight differences in hydrogen-bonding
geometry between the side chain of this residue and S5A of
the cofactor. In addition, in Av1 but not in Cp1 this arginine
residue also hydrogen bonds to the side chain of Asn
98; the
equivalent residue in Cp1 (Phe
89) would not support a
hydrogen-bond interaction.
Four water molecules adjacent to the FeMo cofactor are
conserved in Cp1, Av1 and Kp1, aligned along the Fe3–Fe7
side and within 4.0 A
̊
of cluster sulfurs (Fig. 3; Spatzal
et al.
,
2013; Mayer
et al.
, 1999). In addition, there is also a large water
pool (
30 water molecules) around the homocitrate of the
FeMo cofactor conserved in Cp1, Av1 and Kp1, 13 of which
directly interact with the homocitrate (Fig. 3). This pool
extends between the FeMo cofactor and the P cluster (see
discussion later in this paper).
3.3.2. The P cluster and surrounding environment
.TheAv1
and Kp1 structural studies have established that the P cluster
can adopt at least two distinct conformational states that have
been assigned as the resting (P
N
) and two-electron oxidized
(P
OX
) states (Peters
et al.
, 1997; Mayer
et al.
, 1999). While
these conformations will be described as P
N
and P
OX
,itis
worth noting that these assignments should be considered
tentative and have not been conclusively corroborated by
spectroscopy of the crystalline samples. In the P
N
state, all Fe
atoms in the P cluster are coordinated exclusively by sulfur. Of
note, S1 bridging the two 4Fe–3S partial cubanes is coordi-
nated to six Fe atoms, while the P cluster is coordinated to the
protein through the side-chain sulfurs of six invariant cysteines
from the
-subunits and
-subunits. Upon oxidation, Fe5 and
Fe6 move away from S1, and the coordinating interactions are
replaced by the amide group of Cys
88 and the hydroxyl
group of Ser
188 in Av1 (corresponding to Cys
79 and
Ser
141 in Cp1), respectively (Peters
et al.
, 1997; Mayer
et al.
,
1999). Electron-density analysis and model fitting indicate that
the P cluster in Cp1 corresponds to a
mixture of the P
N
and P
OX
conformations, as
was also observed in the 1.0 A
̊
resolution
Av1 structure (Spatzal
et al.
, 2011; Peters
et
al.
, 1997; Mayer
et al.
, 1999). As shown in
Supplementary Fig. S4, a pronounced
peanut-shaped density is observed around
Fe5 and Fe6 in the 2
F
o
F
c
map, while the
density for all other Fe atoms in the P cluster
displays a well defined spherical shape. This
can be modeled as a mixture of the P
N
and
P
OX
states in a ratio of 4:6 (Mayer
et al.
,
1999; Peters
et al.
, 1997), with Fe5 and Fe6
moving
1.1 A
̊
away from S1 upon oxida-
tion. In the 1.0 A
̊
resolution Av1 structure,
the oxidized state is more populated (
2:8
P
N
:P
OX
) and Fe5/Fe6 appear marginally
further (
0.1 A
̊
) away from S1 than in Cp1
(Supplementary Table S2; Spatzal
et al.
,
2011). The remainder of the P-cluster
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Figure 3
Stereoview of a stick representation of the FeMo cofactor environment in Cp1 (red) and Av1
(cyan). For clarity, only the residues and water molecules participating in polar interactions
with the cofactor are shown. The amino-acid residues are represented by thin lines and waters
are shown as spheres.
structure in Av1 and Cp1 are quite similar, with an overall
r.m.s.d. of 0.05 A
̊
when Fe5 and Fe6 are excluded. The side
chain of Ser
141 in Cp1 also shows two alternative confor-
mations with the hydroxyl-group O atoms positioned 2.1 A
̊
apart, correlated with the alternative conformations of Fe6 in
the P cluster (Fig. 4). Alternative conformations for this
residue are not observed in either Av1 or Kp1. Instead, there
is a water molecule (H
2
O-2) at around 3.3 A
̊
from the
hydroxyl O atom of Ser
188 in Av1 and Kp1, but not in Cp1
(Fig. 4).
The non-S protein ligands in the P
OX
structure, Cys
79 NH
and Ser
141 OH, are ionizable and could potentially contri-
bute to proton-coupled electron transfer and pH-dependence
of the P cluster reduction potential (Lanzilotta
et al.
, 1998).
Remarkably, both residues can be substituted without loss of
function in Av1, in contrast to the other cysteine residues
coordinating the remaining Fe in the P cluster (Dean
et al.
,
1990). In P
OX
, Fe5 is not coplanar with the coordinating amide
group of Cys
79 in any of the three high-resolution MoFe
protein structures, which has been interpreted as an indicator
of a protonated amide N serving as the Fe5 ligand (Mayer
et al.
, 1999; Spatzal
et al.
, 2011). However, a small-molecule
compound with a similarly distorted tetrahedral Fe site
containing a non-coplanar, deprotonated pyrazole nitrogen
ligand has been characterized (Milione
et al.
, 2009). An
additional consideration is that a protonated carboxamido N
would likely be
sp
3
-hybridized to also serve as a metal ligand,
thereby resulting in distortion of the Gly
87–Cys
88 peptide
bond, which is not observed in Cp1, Av1 or
Kp1. As the upper limit of the p
K
a
of a
metal-bound carboxamide is typically
around 4.5 (Noveron
et al.
, 2001; Tyler
et al.
,
2003), it is most likely that a deprotonated
amide N atom coordinates Fe5 in P
OX
in a
distorted, non-coplanar fashion. The side
chain of Ser
141 coordinated to Fe6 in P
OX
(corresponding to Ser
188 in Av1) is also
likely to be deprotonated. This is supported
by the absence of any tetrahedral Fe
complex in the Cambridge Crystallographic
Structure Database (CCDC) coordinating a
protonated organic hydroxyl group (Allen,
2002). Furthermore, the Fe6—O distances in
Cp1 and Av1 (1.93 and 1.91 A
̊
, respectively;
Supplementary Table S3) are well within the
range of the Fe—O bond lengths in tetra-
hedral Fe compounds with deprotonated
organic hydroxyl-group ligands (1.90
0.07 A
̊
for 105 hits with
R
factor
0.075).
Ser
45 (corresponding to Ser
92 in Av1
and Ser
90 in Kp1) is another residue near
the P cluster that may undergo oxidation-
state-induced conformational changes. This
residue is invariant in the group I and group
II MoFe proteins, while either a double
variant (Ser/Ala/Gly) or an invariant Gly
has been found in other groups (Howard
et
al.
, 2013). Two different rotamers for the
side chain of this Ser residue have been
observed in Cp1 and Av1 (Fig. 4). In the
present Cp1 and the 1.0 A
̊
resolution Av1
structure, the hydroxyl O of this residue
faces away from the P cluster (
1
angle of
169.5
in Cp1 and 121.4
in Av1) and
connects to the surface water pool. In the
1.16 A
̊
resolution Av1 structure, where the P
cluster is in the P
N
state, a different rotamer
(
1
angle of 40.7
) is observed, permitting
potential hydrogen-bond formation to S2A
and a conserved water molecule (H
2
O-1).
Considering the location and the confor-
research papers
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Zhang
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Nitrogenase MoFe protein
279
Figure 4
Conformational states of the P cluster in Cp1 and Av1. The P clusters in the Cp1 structure
reflect a superposition of the two conformations corresponding to P
OX
(
a
) and P
N
(
b
) present
at a ratio of 6:4, respectively. The P clusters of the Av1 P
OX
(1.0 A
̊
resolution; PDB entry 3u7q)
and P
N
(1.16 A
̊
resolution; PDB entry 1m1n) conformations are shown in (
c
) and (
d
),
respectively. The backbone atoms of the
-subunits and
-subunits are represented as ribbons
shaded light blue and gray, respectively. The P cluster and coordinated ligand residues as well
as Ser
45 in Cp1 (Ser
188 in Av1) are represented as sticks. The waters are shown as spheres.
C atoms are highlighted in gray, N atoms in blue, O atoms in red, S atoms in yellow and Fe
atoms in orange.
mational alternations observed at this residue, it is reasonable
to propose that Ser
45 may serve as a proton shuttle from the
water pool at the protein surface to the P cluster (Fig. 4).
3.3.3. Hydrogen-bonding network between the P cluster
and the FeMo cofactor
. The tertiary structural alignment of
the MoFe proteins identifies a conserved water tunnel
between the water pool around the homocitrate of the FeMo
cofactor and the P cluster Fe3 in Cp1, Av1 and Kp1, and it may
potentially connect the two metalloclusters through a
hydrogen-bonding network (Spatzal
et al.
, 2011; Mayer
et al.
,
1999). This water tunnel is surrounded by two short and two
long helices: the short helices, involving residues
54–
64 and
residues
77–
83 in Cp1, are approximately parallel to the
tunnel, while the two long helices, corresponding to residues
182–
196 and residues
46–
60 in Cp1, are roughly
perpendicular to the tunnel (Supplementary Fig. S6; Kim
et al.
,
1993). Such conserved structural arrangements may facilitate
electron/proton-transfer process between the two metallo-
clusters (Gray & Winkler, 2005; Markovitch
et al.
, 2008).
3.4. The Fe16 site
Recently, we have reported a 16th Fe (designated Fe16) in
the mononuclear metal-binding site (MMB site) of Av1
between the two
-subunits (Zhang
et al.
, 2013). Fe16 in Av1, a
partially occupied ferrous Fe, exhibits an approximately
octahedral geometry coordinated by the side-chain O atoms of
three Glu/Asp residues, the backbone carbonyl O atom of Arg
and two water molecules. The variation of the three coordi-
nating side-chain residues among all six groups of MoFe
proteins is minor and the variants are also capable of
providing oxygen ligands (Howard
et al.
, 2013). The metal at
the MMB site of Cp1 adopts a similar coordination environ-
ment as in Av1, with a Lys instead of an Arg providing the
carbonyl oxygen ligand (Fig. 5
a
). The site-specific Fe
K
-edge
X-ray absorption near-edge spectrum of Fe16 in Cp1 matches
those of both Av1 and ferrous sulfate well, but not that of
ferric sulfate, indicating that Fe16 in Cp1 is also a ferrous Fe
(Fig. 5
b
; Zhang
et al.
, 2013). The occupancy of Fe16 in Cp1 was
quantified by electron density and by an Fe
K
absorption-edge
jump as described previously using seven sets of MAD data
collected from different Cp1 crystals (Zhang
et al.
, 2013). We
found, using either method, that Fe16 in Cp1 is about half
occupied with little variation between crystals (Supplementary
Table S4). This is a significantly different situation from that
we observed in Av1, where the occupancy of Fe16 essentially
varied from 0 to 1 among the eight sets of MAD data exam-
ined. Another difference in the Fe16 sites between Av1 and
Cp1 is that the alternative conformations observed in the Av1
MMB site are not evident in Cp1 (Fig. 5
a
; Zhang
et al.
, 2013).
This difference could be explained by the presence of Li
+
in
the crystallization solution of Cp1. The average metal–O
distance for Fe16 (2.09 A
̊
) in Cp1 is similar to the average
Li–O distances of Li
+
compounds (2.15
0.08 A
̊
for 197 LiO
6
compounds with
R
factor
0.075) found in the CCDC (Allen,
2002); therefore, a Li or Fe ion may share similar coordination
environment.
Fe16 is approximately 24 and 21 A
̊
away from the P cluster
and the FeMo cofactor, respectively, and may be structurally
coupled to the two metalloclusters through two short
-helices. Lys
61 (Arg
107 in Av1) binds to Fe16 though the
carbonyl O atom and is located at the C-terminal end of a
-subunit helix involving residues
45–
61 in Cp1 (corre-
sponding to residues
92–
108 in Av1), extending between
the FeMo cofactor and the P cluster. Residue Arg
58
(Arg
105 in Av1) in this helix interacts with O6 of the
homocitrate
via
a conserved water molecule (Supplementary
Fig. S5). At the N-terminal end of this helix, Cys
48 binds to
Fe2 and Fe8 in the P cluster. An alternative connection
involves an
-subunit helix (residues
465–
473 in Cp1 and
residues
425–
433 in Av1).
Lys
473 (Lys
433 in Av1) is
about 4.4 A
̊
away from the Fe16
site, while at the other end of this
helix, Lys
466 (Lys
426 in Av1)
and Ile
465 (Ile
425 in Av1)
hydrogen-bond to the C6
carboxyl group of homocitrate
through the side chain or back-
bone amide group, respectively.
The residues mentioned above
are either invariant (Lys
473 and
Cys
48) or single variants
(Lys
466 and Arg
58) with a
similar type of amino acid among
all six groups of nitrogenases
(Howard
et al.
, 2013). In addition,
the three-dimensional structural
arrangements connecting Fe16
and the two metalloclusters are
well conserved in Cp1, Av1 and
Kp1.
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Nitrogenase MoFe protein
ActaCryst.
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Figure 5
Characterization of the MMB site. (
a
)The2
F
o
F
c
electron-density map (light blue) at the MMB site, with
Fe16 and coordinating ligands contoured at 3
. The C atoms are highlighted in gray, N atoms in blue, O
atoms in red and Fe atoms in orange. (
b
) Top: comparison of the refined
f
00
spectrum of Fe16 (red solid
line with red circles) with that of the averaged Fe in the P cluster (black dashed line with white diamonds) of
Cp1 and Fe16 in Av1 (blue dashed line with blue triangles). Bottom: the XAS spectra of ferrous sulfate
heptahydrate (FeSO
4
.7H
2
O, red solid line) and ferric sulfate hydrate [Fe
2
(SO
4
)
3
.
x
H
2
O, black broken line]
(Zhang
et al.
, 2013). No background removal or normalization were applied to the refined
f
00
spectra.
4. Conclusions
In this study, we have characterized the X-ray crystal structure
of Cp1 at atomic resolution and carried out a detailed
comparison with the Av1 structure determined at 1.0 A
̊
resolution. Cp1 and Av1 are representatives of the two distinct
groups of MoFe proteins (
37% sequence identity), differing
primarily by a long insertion in the
-subunit and a deletion in
the
-subunit of Cp1 relative to Av1. Our analysis confirms
that the metal centers, the FeMo cofactor and the P cluster
metallocluster, and the Fe16 mononuclear metal-binding site
are essentially identical in Av1 and Cp1. The structural
arrangements immediately outside the coordination shell of
the FeMo cofactor are highly conserved between the two
MoFe proteins, indicating that such motifs are critical for the
function of the enzyme. More pronounced differences are
observed around the P cluster of the two proteins, suggesting
that the mechanism of nitrogen fixation is less sensitive to
perturbations in this region. Av1 and Cp1 share a conserved
water tunnel and similar secondary-structure elements
between the P cluster and the FeMo cofactor, indicating that
such a structural arrangement is crucial for the interactions
between these two clusters. The most significant differences
are evident in the Fe protein docking surface of the MoFe
protein, which is occluded in Cp1, relative to Av1 and Kp1, by
the long
-subunit sequence insertion. As this loop forms few
contacts with the remainder of the protein, it is plausible that
it rearranges during complex formation to permit similar
interactions during turnover between the MoFe protein and
the Fe protein of different organisms.
Nitrogenase is the only validated biological system capable
of fixing nitrogen; the complexity of the biosynthetic pathway
and the difficulties in preparing synthetic homogeneous
catalysts reinforce the conclusion that dinitrogen reduction
under physiological conditions is a demanding reaction to
achieve. Giving the challenges in developing a successful
catalyst like nitrogenase, it is likely there are significant
restrictions in the active-site variation that can be tolerated. A
comparison of Cp1 with Av1 supports this view; although the
overall sequence identity is
37%, the metal centers are
essentially superimposable in these
1A
̊
resolution structures
and the protein environment surrounding the active-site FeMo
cofactor is highly conserved both in terms of sequence and
structure. While the basic framework is maintained, greater
variation is tolerated surrounding the P cluster and at the Fe
protein docking surface, suggesting that intermolecular and
intramolecular electron and proton transfers to the active site
may be less sensitive to variations in sequence and structure.
We thank Dr J. B. Howard for contributions to this project.
The work was supported by NIH grant GM45162 to DCR, a
National Sciences and Engineering Research Council of
Canada postdoctoral fellowship and Caltech Center for
Environmental Microbial Interactions fellowships to L-MZ
and a National Science Foundation Graduate Research
Fellowship (grant DGE-1144469 to CNM). We thank the staff
at beamline 12-2, Stanford Synchrotron Radiation Light-
source (SSRL) for their assistance. SSRL is operated for the
DOE and supported by its OBER and by the NIH, NIGMS
(P41GM103393) and the NCRR (P41RR001209). We
acknowledge the Gordon and Betty Moore Foundation, the
Beckman Institute and the Sanofi–Aventis Bioengineering
Research Program at Caltech for their generous support of the
Molecular Observatory at Caltech.
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